Phagemid for antibody screening

ABSTRACT

A phagemid has been constructed that expresses an antibody fused to coliphage pIII protein. The phagemid is suitable for selecting specific antibodies from large gene libraries with small quantities of antigen. The antibody-pIII gene can be strongly repressed, so that it allows antibody libraries to be amplified without the danger of deletion mutants predominating. After induction, large quantities of the fusion protein may be expressed.

This application is a continuation of application Ser. No. 09/645,436,filed on Aug. 25, 2000, now U.S. Pat. No. 6,387,627, which is adivisional of 09/414,005, filed on Oct. 7, 1999, now U.S. Pat. No.6,127,132, which is a divisional of 09/135,002, filed on Aug. 17, 1998,now U.S. Pat. No. 5,985,588, which is a continuation of 07/982,743 filedon May 10, 1993, now U.S. Pat. No. 5,849,500, and for which priority isclaimed under 35 U.S.C. 120; application Ser. No. 07/982,743 is thenational phase of PCT International Application No. PCT/EP92/01524 filedon Jul. 6, 1992 under 35 U.S.C. 371. The entire contents of each of theabove-identified applications are hereby incorporated by reference. Thisapplication also claims priority of Application No. P41 22599.6 filed inGermany on Jul. 8, 1991 under 35 U.S.C. 119.

The present invention concerns phagemids for the selection of specificantibodies from large recombinant libraries, the production of thesephagemids and their use to select specific antibodies from largerecombinant libraries using small amounts of antigen.

Plasmid and phage antibody libraries have been established in E. colifrom PCR amplified immunoglobulin families following immunization.Recombinant antibodies to immunogens were selected by an ELISA assay ofthe bacterial supernatant from isolated bacterial colonies (Ward, E. S.,Güssow, D., Griffiths, A. D., Jones, P. T. & Winter, G.: Bindingactivities of a repertoire of single immunoglobulin variable domainssecreted from Escherichia coli. Nature 341 (1989) 544-546) or byscreening nitrocellulose plaque lift-offs of bacterial colonies forreactivity to the radioactively labeled immunogen (Huse, W. D., Sastry,L., Iverson, S. A., Kang, A. S., Alting-Mees, M., Burton, D. R.,Benkovic, S. J. and Lerner, R. A.: Generation of a large combinatoriallibrary of the immunoglobin repertoire in phage lambda. Science 246(1989) 1275-1281). However, for the selection of specific antibodiesfrom randomly combined light and heavy chain libraries of non-immunizedanimals that do not contain a preponderance of antibodies to aparticular antigen, a procedure is required for screening millions ofantibody producing bacteria.

A possible way to screen a broad range of antibodies is to attachrecombinant antibodies to the surface of bacteria or bacteriophages sothat they can then be rapidly selected by antigens bound to a solidphase. Given the difficulties of targeting proteins to the cell surfaceof bacteria, an attractive candidate in view of its small size andrelatively simple genetic make-up is the M13 family of filamentousbacteriophages (for reviews see Webster, R. E. and Lopez, J. in “VirusStructure and Assembly” ed. S. Casjens, publ. Jones and Bartlett Inc.,Boston/Portala Valley, USA, 1985; Day, L. A., Marzec, C. J., Reisberg,S. A. and Casadevall, A.: DNA packaging in filamentous bacteriophages.Ann. Rev. Biophys. Biophys. Chem. 17 (1988) 509-539).

The product of gene III (pIII) is a relatively flexible and accessiblemolecule composed of two functional domains; an amino-terminal domainthat binds to the F pilus of male bacteria during infection and acarboxy-terminal domain buried within the virion that is important formorphogenesis. Peptides can be inserted between the two domains of pIII(Smith, G. P.: Filamentous fusion phage: novel expression vectors thatdisplay cloned antigens on the virion surface. Science 228 (1985)1315-1317) or near the N-terminus (Parmley, S. F. and Smith, G. P.:Antibody-selectable filamentous fd phage vectors: affinity purificationof target genes. Gene, 73 (1988) 305-318) without destroying itsfunctions in morphogenesis and infection. After much pioneering work onthe use of pIII in fd phages for carrying foreign peptides, Parmely andSmith (1988, a.a.O.) showed that peptide epitopes inserted at theaminoterminal end could bind phages to immobilized antibodies. As aconsequence of this work it has been possible to generate peptidelibraries that can be screened for binding to ligands and antibodies(Scott, J. K. and Smith, G. P.: Searching for peptide ligands with anepitope library. Science 249 (1990) 386-390; Devlin, J. J., Panganiban,L. C. and Devlin, P. E.: Random peptide libraries; A source of specificprotein binding molecules. Science 249 (1990) 404-406; Cwirla, S. E.,Peters. E. A., Barrett, R. W. and Dower, W. J. Peptides on phage, a vastlibrary of peptides for identifying ligands.: Proc. Natl. Acad. Sci.USA, 87 (1990) 6378-6382).

McCafferty, J., Griffiths, A. D., Winter, G. and Chiswell, D. J.: Phageantibodies: filamentous phage displaying antibody variable domains.Nature, 348 (1990) 552-554 reported the assembly of an antibody-pIIIfusion protein into an fd phage with a Tet^(R) gene after insertingantibody DNA into the 5′ end of gene III. The phage remained infectiousand was able to be enriched by affinity chromatography. However, fusionphage have been shown to be mainly useful for displaying relativelysmall inserts, probably, because the large inserts have an adverseeffect on the infectivity function of pIII (Parmlee and Smith, 1988,a.a.O). There is a large risk, therefore, that phage libraries willquickly become dominated by deletion mutants after libraryamplification.

Thus the technical problem underlying the present invention is toprovide a more efficient mean for screening antibody libraries inbacteria.

This problem is solved by providing a phagemid according to claim 1 thatexpresses a functional antibody-pIII fusion protein. Preferably theantibody is a single-chain antibody. DNA coding for an antibody-pIIIfusion protein, preferably a single-chain antibody-pIII fusion protein,was incorporated into a phagemid. A major advantage of the phagemidsystem of this invention over McCafferty et al. (see above) is that itcan be propagated as a plasmid and is not under any selection pressureto remove antibody DNA, since the expression of the fusion protein istightly repressed. This is particularly important during theamplification of antibody libraries when faster proliferating deletionmutants could quickly dominate. The phagemid DNA, being less than halfthe size of the above phage DNA, also transform bacteria moreefficiently. Moreover, in contrast to the above mentioned phage system,large quantities of the smaller phagemid DNA are produced and largeamounts of antibody protein are available after induction, therebygreatly facilitating its analysis. Expression of the antibody-pIIIfusion protein, preferably the single-chain antibody-pIII fusionprotein, using the pSEX phagemid (see below) and its packaging intoviral particles greatly facilitate the establishment of bacterialsystems for the isolation of high affinity antibodies. Millions ofantibody-producing clones from antibody libraries can now be rapidlyscreened by binding to immobilized antigen. A further advantage overconventional screening methods is that only small amounts of antigen arerequired, an important factor when the supply of a rare protein islimited. This system also offers the possibility of screening randomlymutated antibodies in order to increase their binding affinities. Theprocedure could be repeated many times until the desired specificity isachieved. It is now feasible for the first time to carry out large scaledifferential screening analyses of related cells and organisms. Asubstractive selection, e.g., using normal and neoplastic cells could beused to identify tumor associated antigens. The phagemid system alsoproves to be extremely useful for investigating the component ofmolecular interactions e.g. by selecting antibodies that inhibit ligandreceptor binding.

Furthermore, the system of this invention is mostly useful forpresenting other proteins or peptides at the surfaces of phagemid viralparticles. For this purpose the DNA encoding the antibody has to bereplaced with DNA of the desired polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a restriction map of the phagemid pSEX.

FIG. 1B (SEQ ID NOS: 7-15) describes the ribosome binding site (RBS),leader sequence of pectate lyase, tag linker and the PCR primers forpIII.

FIG. 1C (SEQ ID NOS: 16-17) describes an alternative tag-linkersequence.

FIGS. 2A-2B show the results of a Western blot analysis foridentification of the antibody-pIII fusion protein.

FIG. 3 shows the results of gel electrophoresis of circular singlestranded pSEX.

The following Examples illustrate but not restrict the presentinvention.

EXAMPLES Example 1

Construction of Phagemid (pSEX)

DNA coding for a single chain antibody (scAb) and pIII were cloned intopUC119 after insertion of a specific set of restriction sites and aprotease sensitive joining sequence into the multiple cloning site. TheAb-DNA coded for the heavy and light chain variable domains of ahumanised Ab against hen egg white lysozyme derived from theanti-lysozyme Ab D1.3 (Amit et al., Science 233, p. 747-754, 1986;Verhoeyen, M. et al. Science 239 p. 1534-1536, 1988). These domains werejoined by an eighteen amino acid linker sequence containing the epitopefor the monoclonal Ab YOL1/34 (Breitling, F. and Little, M.:Carboxy-terminal regions on the surface of tubulin and microtubules.Epitope locations of YOL1/34, DM1A and DM1B. J. Mol. Biol. 189 (1986),367-370), thus enabling the Ab to be identified. To provide a moreflexible junction to pIII, the 3′ end of the light chain DNA wasmodified by the addition of nucleotides coding for the first six aminoacids of the human kappa constant domain followed by a BamHI restrictionsite. pIII DNA was amplified from the bacteriophage M13 using primerscorresponding to the 5′ and 3′ ends of gene III. The Ab-pIII-DNA wasthen cloned into a phagemid of the pDS family that contains a coliphageT7 promoter combined with two lac operators (Bujard, H., Gentz, R.,Lancer, M., Stüber, D., Müller, H.-M., Ibrahim, I., Häuptle, M.-T. andDobberstein, B.: A T5 promoter-based transcription-translation systemfor the analysis of proteins in vitro and in vivo. Methods Enzymol. 155(1987) 416-433; Lancer, M. and Bujard, H.: Promoters determine theefficiency of repressor action. Proc. Natl. Acad. Sci. USA 85 (1988)8973-8977); Müller, H.-M. Ph.D. thesis, Univ. of Heidelberg, 1989). In afinal step, DNA coding for the leader sequence of the bacterial enzymepectate lyase was ligated to the 5′ end of the Ab-DNA resulting in thephagemid pSEX (FIG. 1a). The leader, linker and PCR primer sequences areshown in FIG. 1b. An alternative linker sequence (FIG. 1c) with theYOL1/34 epitope placed at the end of the linker containing a usefulrestriction site for the insertion of Ab libraries was also employed.Although both of these tag-linkers contain a significant number ofacidic residues, they appeared to have no effect on the production offunctional scAbs when compared to scAbs with linkers composed only ofthe neutral amino acids glycine and serine.

Example 2

Expression of Antibody-pIII Fusion Protein

To test whether the completed phagemid vector was able to express thefull length fusion protein, 100 μM IPTG was added to a log phase cultureof E. coli transformed with pSEX. The culture showed a marked decline inits rate of growth compared to the control indicating a significantsynthesis of phagemid encoded protein. On Western blot analysis, theantibody-pIII construct was identified by three antibodies; a monoclonalantibody to part of the linker sequence (EEGEFSEAR) (SEQ ID NO: 10,residues 3-II) and two anti-peptide rabbit sera against N-terminalsequences of the heavy and light chains (QVQLQQSGGG (SEQ ID NO: 1) andDIQMTQSPSS (SEQ ID NO: 2), respectively). It migrated with an apparentmolecular weight of 93 kd (FIG. 2). The large size of the fusion protein(predicted: Mr 68100) is most probably due to the pIII component (Mr42100) that migrates with an apparent molecular weight of approximately55000-70000 kd (Goldsmith, M. E. and Konigsberg, W. H.: Adsorptionprotein of the bacteriophage fd: isolation, molecular properties andlocation in the virus. Biochemistry, 16 (1977) 2686-2694). Partialproteolysis of the fusion protein was indicated by the presence of someminor bands of lower molecular weight that were identically stained byall three antibodies.

Cell fractionation showed that the protein was present in thecytoplasmic and membrane fractions but not in the periplasm and culturesupernatant (FIG. 2, lanes 3-6) in contrast to the antibody componentalone without pIII that was secreted into the periplasm and medium (datanot shown). This was not surprising since pIII is assembled onto phageparticles from the inner bacterial membrane, a process that appears tobe dependent only on the C-terminal domain. Deletion mutants of pIIIwithout this domain pass into the periplasm without becoming attached tothe cytoplasmic membrane (Boeke, J. D. and Model, P.: A prokaryoticmembrane anchor sequence: carboxyl terminus of bacteriophage f1 gene IIIprotein retained in the membrane. Proc. Natl. Acad. Sci. USA 79 (1982)5200-5204) and normal phage particles are not assembled (Crissman, J. W.and Smith, G. P.: Gene III protein of filamentous phages: evidence for acarboxy-terminal domain with a role in morphogenesis.: Virology, 132(1984) 445-455). The anchor sequence is probably a hydrophobic stretchof twenty three amino acids at the carboxy-terminus (Davis, N. G.,Boeke, S. and Model, P.: Fine structure of a membrane anchor domain. J.Mol. Biol. 181 (1985) 111-121).

The ability of the fusion protein to bind antigen was investigated bypassing the triton soluble fraction over a column of lysozyme bound tosepharose. Western blots of the unbound material and the fractionsobtained after thoroughly washing and eluting with 0.05 M diethylamineshowed that the full length fusion protein was indeed specificallyretained on the lysozyme column (FIG. 2, lanes 7-12).

Example 3

Packaging of the pSEX Phagemid

To determine whether the phagemid expression vector could be packaged,E. coli containing pSEX were multiply infected with phage fd. IPTG wasnot added since it was found to have an inhibitory effect on phagemidpackaging. A similar finding was recently reported by Bass et al.,Proteins 8, S. 309-314 (1990) who constructed a phagemid that expressesa fusion protein of human growth hormone and the C-terminal domain ofpIII. Examination of Ab-pIII production with and without IPTG afteradding phage fd showed that the phage alone was able to induceexpression (FIG. 3). A possible explanation is that one of the phagegene products interferes with the binding of lac repressor to theoperator. Alternatively, phage proteins binding to the intergenic regionmight affect the topology of the phagemid and cause the release of thelac repressor. Whatever the reason, we have found that moving theintergenic region 10³ nucleotides to the other side of the bla gene hasno effect on this phenomenon (data not shown).

Agarose gel electrophoresis of the DNA from virus particles secretedinto the medium showed, in addition to the single stranded DNA of fd, alarger quantity of smaller DNA that was compatible in size with singlestranded pSEX. Further proof of phagemid packaging and the production ofinfectious particles was shown by infecting E.coli with the secretedvirus particles. 10¹⁰/ml AmpR colonies of E.coli were obtained incomparison to 3×10⁹ pfu.

To determine whether the packaged phagemid had incorporated theantibody-pIII fusion protein, 90 μl culture supernatant containing 5×10⁸packaged phagemids determined as Amp transducing units was mixed with a1000-fold excess of wild type fd phage and passed over a column ofimmobilized lysozyme. After thoroughly washing with ten bed volumes ofPBS, 1M NaCl and 0.5 M NaCl in 0.1 M NaHCO₃ at pH 8.3, respectively, thephagemid particles were eluted with 0.05 M diethylamine. The eluate wasneutralized with 0.5 M NaH₂PO₄ and assayed for the number of phages andpackaged phagemids (Table). A specific enrichment of up to 121-fold wasachieved, thus demonstrating the incorporation of functionalantibody-pIII constructs into phagemid particles. The binding propertiesof the phagemid particles might be further increased by using a pIIIdeletion mutant for packaging. This would ensure that only thosephagemids coding for functional fusion proteins would be packaged andall five pIII proteins on a phagemid particle would be fused toantibodies.

DESCRIPTION OF THE FIGURES

FIG. 1 Construction of pSEX, a phagemid for antibody screening

VH and VL are heavy and light chain variable domains, respectively, ofan anti-lysozyme Ab.

a) Construction.

To provide the necessary restriction sites, the oligo-nucleotides5′GCTGAATTCGGATCCATAGGGCCCTCTAGAGTCGAC3′ (SEQ ID NO: 3) and5′AATTGTCGACTCTAGAGGGCCCTATGGATCCGAATTCAGCTGCA3′ (SEQ ID NO: 4) were 5′phosphorylated, hybridized and ligated to pUC119 that had been cleavedwith PstI and EcoRI and dephosphorylated. In an optional step to createa protease sensitive sequence, the hybridized oligonucleotides5′GATCCAAAGATATCAGAGGGCC3′ (SEQ ID NO: 5) and 5′ CTCTGATATCTTTG3′ (SEQID NO: 6) were inserted between the BamHI and ApaI sites of the firstset of oligonucleotides. scAB-DNA was then inserted between the PstI andBamHI followed by the blunt end ligation of pIII DNA after cleaving thephagemid with ApaI and treating with T4 DNA polymerase to remove 3′overhanging ends. pSEX was constructed by combining the multiple cloningsite of pUHE24-2 with the closely related phagemid pDS31-1 that containsan additional f1 intergenic region (Bujard et al., 1987, see above,;Müller, 1989, see above). The pDS31-1 sequence extends from Xho1anticlockwise to a HindIII site (in parentheses) that was lost after ablunt end ligation. pUHE24-2 is essentially identical to pDS6 (Bujard etal., 1987) with a coliphage T7 promoter combined with two lac operatorsand a ribosome binding site (PA1/04/03, Lancer and Bujard, 1988, seeabove; Lanzer, 1988, see above). The resulting phagemid was cleaved withHindIII and the 5′overhangig ends were filled up with Klenow fragment.After a further digestion with PstI, the PstI-HincII Ab-pIII-DNAfragment was inserted into the phagemid. In a final step, synthetic DNAcoding for the leader sequence of the bacterial enzyme pectate lyase andfor the first four amino acids of the heavy chain was inserted betweenthe NcoI and PstI restriction sites. pUHE plasmids were propagated inE.coli 71-18 with the plasmid pDM1 that expresses lac repressor, pUCplasmids were propagated DH5α and the antibody-pIII fusion protein wasexpressed in JM101.

b) Sequence of the ribosome binding site (RBS), leader sequence ofpectate lyase, tag-linker and the PCR primers for pIII. Underlined aminoacids indicate the epitope for YOL1/34. The following amino acids in thelinker sequence are a continuation of the α-tubulin sequence. (NOTE SEQID NOS: 7-15).

c) Alternative tag-linker sequence. Underlined amino acids indicate theepitope for YOL11/34. The preceding linker amino acids are acontinuation of Ab sequence into the constant domain. (NOTE SEQ ID NOS:16-17)

FIG. 2 Inducibility, cellular localization and antigen binding of theantibody-pIII fusion protein analyzed by gel electrophoresis on 8%polyacrylamide gels and Western blotting Lanes 1 and 2: Total cellsafter 1 h induction with 100 μM IPTG (1) or without IPTG (2). Lanes 3-6:Cell fractionation; 3: culture supernatant, 4: periplasmic enrichedfraction, 5: soluble cytoplasmic fraction, 6: 1% triton extract. Lanes7-12: Lysozyme affinity chromatography of the 1% triton extract frominduced and non-induced cells, 7: effluent (+IPTG), 8: effluent (−IPTG),9: last wash (+IPTG), 10: last wash (−IPTG), 11: eluate (+IPTG), 12:eluate (−IPTG).

Lanes 1-6 were stained using the monoclonal antibody YOL1/34 (Serotec,Oxford, U.K.) and lanes 7-12 using an antiserum to the N-terminalsequence of the light chain.

Method:

Antisera to the heavy and light chains were obtained by the subcutaneousinjection of rabbits with the amino-terminal peptides QVQLQQSGGG (AC)(SEQ ID NO: 1) and DIQMTQSPSS (AC) (SEQ ID NO:2), respectively, coupledto keyhole limpet heamocyanin. To investigate the expression of thefusion protein, the pelleted bacteria of IPTG-induced cultures wereresuspended in 30 mM Tris/HCl, pH 8.0 containing 20% sucrose. 1 mM EDTA,1 mg/ml chick lysozyme and incubated for 10 min on ice. Aftercentrifuging for 1 min at 15000 g, the supernatant containing theperiplasmic proteins was collected and the pellet was sonicated in 0.1 MTris/HCl pH 8.0. The soluble cytosolic fraction was decanted aftercentrifuging for 5 mm at 15000 g and the resuspended pellet wasincubated in 1% Triton X100 to obtain the membrane bound fraction. Allthe fractions were analyzed for p-lactamase activity according toPlückthun, A. and Knowles, J. R.: The consequences of stepwise deletionsfrom the signal-processing site for β-lactamase. J. Biol. Chem. 262(1987) 3951-3957 to check the efficiency of the fractionation procedure.The triton soluble fraction was diluted a 100-fold with PBS beforeapplying to affinity columns. For affinity chromatography, chicklysozyme (Boehringer, Mannheim, FRG) was coupled to cyanogen bromideactivated Sepharose (Pharmacia) according to the instructions of themanufacturer. The lysozyme-Sepharose was incubated for 20 mm at roomtemperature with the extracts and poured into columns that weresubsequently washed with ten bed volumes of PBS, 1M NaCl and 0.5M in0.1M NaHCO₃ at pH 8.3, respectively, before eluting with 0.05Mdiethylamine. All the fractions were precipitated with trichloraceticacid (final concentration 20%) and resolved on SDS polyacrylamide gels(Laemmli, U.K.: Cleavage of structural proteins during the assembly ofthe head of the bacteriophage T4. Nature 227 (1970) 680-685)- Westernblots were performed according to Towbin, H. Staehelin, T. and Gordon,I.: Electrophoretic transfer of protein from polyacrylamide gels tonitro-cellulose sheets: procedures and some applications (1979) Proc.Natl. Acad. Sci. USA 76, 4350-4354, using second antibodies coupled tohorseradish peroxidase with diaminobenzidene as substrate.

FIG. 3 Gel electrophoresis of circular single stranded pSEX

Lane 1: fd, lane 2: control phagemid pUHE31-1 with fd, lane 3: pSEX1with fd.

DNA from fd virions and packaged phagemid particles was applied to 0.8%agarose gels in 1×TBE according to Sambrook, J., Fritsch, E. F. andManiatis, T. in Molecular Cloning: A Laboratory Manual, 2nd. ed. ColdSpring Harbor Laboratory (1989) and stained with ethidium bromide.

Method:

For the preparation of packaged phagemids, E. coli JM101 containingpSEX1 was plated onto M9 minimal medium and incubated for 30 h at 37° C.2 ml of the same medium was inoculated with one of the colonies andincubated at 37° C. with vigorous aeration until it reached an opticaldensity of about 0.2 at 600 nm. 0.5 ml of LB medium and a 10-fold excessof phage fd were then added to the culture and it was incubated for afurther 3 h at 37° C. After carefully centrifuging twice at 15000 g for5 min at room temperature, the supernatant was adjusted to a finalconcentration of 4% polyethylene glycol (Serva PEG 6000) and 0.5M NaCland left to stand overnight at 4° C. The phagemids were sedimented bycentrifuging at 15000 g for 20 min at room temperature and suspended in200 μl of a Tris-EDTA buffer, pH 7.5. Phagemid DNA was prepared byshaking with one volume of phenol for ten minutes followed by treatmentwith chloroform-isopropanol and precipitated with isopropanol (Sambrook,J., Fritsch, E. F. and Maniatis, T. in Molecular Cloning: A LaboratoryManual, 2nd. ed. Cold Spring Harbor Laboratory (1989)).

TABLE Specific enrichment of packaged phagemids on an antigen affinitycolum Amp^(r) Colonies Total pfu Total Plated Plated Excesss EnrichmentVolume (ml) Volume (μl) Number Total Volume (μl) Number Totalpfu/Amp^(r) Factor Applied 10 10⁻³ 51 5.1 × 10⁸ 10⁻⁶ 45 4.5 × 10¹¹ 882 —Eluted 1.1 10⁻¹ 26 2.9 × 10⁵ 10⁻² 19 2.1 × 10⁶ 7.3 121

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 17(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 10 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide   (iii) HYPOTHETICAL: NO      (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:           (A) NAME/KEY: Peptide          (B) LOCATION: 1..10           (D) OTHER INFORMATION: #/label= peptide                /note= #“N-terminal sequence of heavy chain                variable #domain of scAb to hen egg white                lysozyme.”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:Gln Val Gln Leu Gln Gln Ser Gly Gly Gly 1               5   #                10 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10 amino #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide      (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:           (A) NAME/KEY: Peptide          (B) LOCATION: 1..10           (D) OTHER INFORMATION: #/label= peptide                /note= #“N-terminal sequence of light chain                variable #region of scAb to hen egg white lysozyme”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 1               5   #                10 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 36 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: /label=  #linker top strand    (ii) MOLECULE TYPE: DNA    (iii) HYPOTHETICAL: NO     (ix) FEATURE:          (A) NAME/KEY: -           (B) LOCATION: 1..36    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:GCTGAATTCG GATCCATAGG GCCCTCTAGA GTCGAC       #                  #       36 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 44 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA    (ix) FEATURE:           (A) NAME/KEY: -          (B) LOCATION: 1..44           (D) OTHER INFORMATION: #/label= linker bottom strand     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #4: AATTGTCGAC TCTAGAGGGC CCTATGGATC CGAATTCAGC TGCA   #                   # 44 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 22 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA    (ix) FEATURE:           (A) NAME/KEY: -          (B) LOCATION: 1..22           (D) OTHER INFORMATION: #/label= linker                /note=  #“top strand of linker encoding a               protease-sen #sitive                sequence”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5:GATCCAAAGA TATCAGAGGG CC            #                  #                 22 (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 14 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA    (ix) FEATURE:           (A) NAME/KEY: -          (B) LOCATION: 1..14           (D) OTHER INFORMATION: #/label= linker                /note= #“bottom strand of linker encoding                protease-sen#sitive sequence”     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6:CTCTGATATC TTTG               #                   #                  #     14 (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 110 base #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: both          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA    (ix) FEATURE:           (A) NAME/KEY: -          (B) LOCATION: 1..110           (D) OTHER INFORMATION: #/label= fragment                /note= #“EcoRI - PstI fragment of pSEX (nucleotides                86 - 1#96), see Fig. 1”     (ix) FEATURE:           (A) NAME/KEY: CDS          (B) LOCATION: 30..110           (D) OTHER INFORMATION: #/product= “pelB leader sequence”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7:GAATTCATTA AAGAGGAGAA ATTAACTCC ATG AAA TAC CTC TTG# CCT ACG GCA       53                    #              Met Lys T#yr Leu Leu Pro Thr Ala                    #                1  #             5 GCC GCT GGC TTG CTG CTG CTG GCA GCT CAG CC#G GCG ATG GCG CAA GTT      101Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pr #o Ala Met Ala Gln Val     10              #     15              #     20CAG CTG CAG               #                   #                  #        110 Gln Leu Gln  25 (2) INFORMATION FOR SEQ ID NO: 8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 amino #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8:Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gl #y Leu Leu Leu Leu Ala  1               5  #                 10  #                 15Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gl #n              20     #             25 (2) INFORMATION FOR SEQ ID NO: 9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 66 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: both          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA    (ix) FEATURE:           (A) NAME/KEY: -          (B) LOCATION: 1..66           (D) OTHER INFORMATION: #/label= fragment                /note= #“tag-linker/scAb boundary in plasmid pSEX                (nucleotides# 523 - 589), see Fig. 1”     (ix) FEATURE:           (A) NAME/KEY: CDS          (B) LOCATION: 1..66     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #9: TCC TCA GAA GAA GGT GAA TTC TCA GAA GCT CG#T GAA GAT ATG GCT GCA       48Ser Ser Glu Glu Gly Glu Phe Ser Glu Ala Ar #g Glu Asp Met Ala Ala  1               5  #                 10  #                 15CTT GAG AAA GGT GAT ATC          #                   #                  #  66 Leu Glu Lys Gly Asp Ile              20(2) INFORMATION FOR SEQ ID NO: 10:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 22 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #10:Ser Ser Glu Glu Gly Glu Phe Ser Glu Ala Ar #g Glu Asp Met Ala Ala  1               5  #                 10  #                 15Leu Glu Lys Gly Asp Ile              20(2) INFORMATION FOR SEQ ID NO: 11:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 60 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: both           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA     (ix) FEATURE:           (A) NAME/KEY: -          (B) LOCATION: 1..60           (D) OTHER INFORMATION: #/label= fragment                /note= #“portion of pSEX plasmid (nucleotides 895                -               954) at  #scAb/gene III boundary, see Fig. 1”    (ix) FEATURE:           (A) NAME/KEY: CDS          (B) LOCATION: 1..60     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #11: AAA CGT ACG GTA GCA GCT CCT GGA TCC AAA GA#T ATC AGA GCT GAA ACT       48Lys Arg Thr Val Ala Ala Pro Gly Ser Lys As #p Ile Arg Ala Glu Thr  1               5  #                 10  #                 15GTT GAA AGT TGT             #                   #                  #       60 Val Glu Ser Cys              20(2) INFORMATION FOR SEQ ID NO: 12:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 20 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #12:Lys Arg Thr Val Ala Ala Pro Gly Ser Lys As #p Ile Arg Ala Glu Thr  1               5  #                 10  #                 15Val Glu Ser Cys              20 (2) INFORMATION FOR SEQ ID NO: 13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 36 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: both          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA    (ix) FEATURE:           (A) NAME/KEY: -          (B) LOCATION: 1..36           (D) OTHER INFORMATION: #/label= fragment                /note= #“end of fusion protein-encoding insert in                pSEX (nuc#leotides 2137 to HindIII site), see Fig.                1”    (ix) FEATURE:           (A) NAME/KEY: CDS          (B) LOCATION: 1..15           (D) OTHER INFORMATION: #/product= “end of gene III portion                of scAb/g#ene III fusion protein”     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #13:CGT AAT AAG GAG TCT TAATGACTCT AGAGTCAGCT T   #                  #       36 Arg Asn Lys Glu Ser   1               5(2) INFORMATION FOR SEQ ID NO: 14:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 5 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #14: Arg Asn Lys Glu Ser  1               5 (2) INFORMATION FOR SEQ ID NO: 15:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 18 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA    (ix) FEATURE:           (A) NAME/KEY: -          (B) LOCATION: 1..18           (D) OTHER INFORMATION: #/label= primer                /note=  #“backward primer, see Fig. 1”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #15:TCATTAAGAC TCCTTATT              #                   #                  #  18 (2) INFORMATION FOR SEQ ID NO: 16:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 63 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: both          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA    (ix) FEATURE:           (A) NAME/KEY: -          (B) LOCATION: 1..63           (D) OTHER INFORMATION: #/label= linker                /note= #“alternative tag-linker sequence, see Fig.                1(c)”    (ix) FEATURE:           (A) NAME/KEY: CDS          (B) LOCATION: 1..63     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #16: TCA GGG AGT GCA TCC GCC CCA AAG CTT GAA GA#A GGT GAA TTC TCA GAA       48Ser Gly Ser Ala Ser Ala Pro Lys Leu Glu Gl #u Gly Glu Phe Ser Glu  1               5  #                 10  #                 15GCG CGC GAA GAT ATC            #                   #                  #    63 Ala Arg Glu Asp Ile              20(2) INFORMATION FOR SEQ ID NO: 17:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 21 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #17:Ser Gly Ser Ala Ser Ala Pro Lys Leu Glu Gl #u Gly Glu Phe Ser Glu  1               5  #                 10  #                 15Ala Arg Glu Asp Ile              20

What is claimed is:
 1. A phagemid comprising DNA encoding a polypeptide-coliphage pIII fusion protein, wherein said fusion protein comprises a polypeptide and a coliphage pIII polypeptide and wherein said coliphage pIII polypeptide comprises contiguous amino and carboxy domains of a coliphage pIII protein.
 2. A phagemid according to claim 1 wherein said polypeptide-coliphage pIII fusion protein contains a protease-sensitive region between the polypeptide and the coliphage pIII polypeptide.
 3. A phagemid according claim 1 wherein said phagemid further comprises expression control elements upstream of said DNA and further encodes at least one selectable marker.
 4. A phagemid according to claim 1 wherein said phagemid is packaged into viral particles.
 5. A process for the production of a phagemid according to claim 1, comprising the step of incorporating the DNA encoding said polypeptide-coliphage pIII fusion protein into a phagemid vector.
 6. The process according to claim 5 further comprising inserting a protease-sensitive site between the DNA encoding the polypeptide and the DNA encoding the coliphage pIII polypeptide.
 7. A library of phagemids wherein said phagemids are as defined in any one of claims 1 or 2-4 and wherein said phagemid library contains a plurality of members encoding different polypeptides.
 8. A library of phagemids wherein said phagemids are packaged into viral particles according to claim 4 and wherein said packaged phagemid library contains a plurality of members encoding different polypeptides.
 9. A method of screening for polypeptides from a library according to claim 8, comprising exposing said library to a ligand, and selecting those packaged phagemids that bind to the light.
 10. A method according to claim 9 further comprising the production of a polypeptide identified following said screening of said library or a portion of said library.
 11. A method of presenting a polypeptide at a surface of a phagemid viral particle, comprising producing said phagemid viral particle, wherein said phagemid viral particle comprises a DNA sequence encoding said polypeptide fused to a DNA sequence encoding a coliphage pIII polypeptide comprising contiguous amino and carboxy domains of a coliphage pIII protein. 